Vaccination of Rhesus Macaques with an attenuated CMV virus carrying SIV proteins (RhCMV-SIV) led to an unprecedented durable control and clearance of SIV in 50% of vaccinated Macaques, a positive outcome predicted by the magnitude of CD8 T cell responses during vaccination and effector T cell responses in lymph nodes after infection. RhCMV-SIV vaccination broke natural immunodominance and led to broad CD4 T cells and CD8 responses covering many areas of SIV antigens. Surprisingly most SIV-specific CD8 T cells responses elicited by this vaccine were restricted by MHC-II. Whereas the results are among the most promising to date in the HIV vaccine field the use of CMV vectors for HIV vaccination raise health concerns. The first event required to prime T cell responses is the presentation of MHC-I- and MHC-II-bound peptides by antigen presenting cells (APC) to T cells. These peptides come from the multistep intracellular degradation of proteins by the antigen processing machinery. Considering the capacity of WT CMV to alter MHC-peptide presentation we propose that attenuated CMV expressing HIV proteins (HCMV-HIV) creates unique conditions for processing and presentation of HIV epitopes leading to broad unconventional T cell responses, and that these conditions can be replicated without the use of CMV by transiently manipulating the antigen processing machinery during the delivery of the immunogen in APC. To unveil these mechanisms of protein degradation and epitope presentation in the presence of attenuated HCMV-HIV we have developed throughput assays to measure antigen processing activities in primary cells, mass spectrometry-based assays to follow the degradation of proteins or virions in the two cellular compartments where the virus may enter: cytosol and endo-lysosomes, and methodologies to identify intracellular epitope precursors and MHC-bound peptides in HIV-infected cells. We showed that variations in the levels of peptidase activities among cell subsets play a critical role in shaping the lengths and kinetics of peptides produced for epitope presentation. Using a combination of biochemical, computational, in vitro and in vivo immunological approaches we propose to 1) Determine the effect of attenuated HCMV-HIV on HIV protein degradation in target cell subsets, 2) Assess epitope presentation and priming of T cell responses in the context of attenuated HCMV-HIV infection, and 3) Design and test a CMV-free antigen delivery system leading to broad HIV-specific T cell responses. This project builds on a multidisciplinary collaborative approach between the PI, Dr Heckerman for computational analysis of HIV degradation products, Dr Picker for HCMV expertise in CMV, Dr Moris for in vitro priming assays of HIV-specific T cell responses, Dr Walker for HIV-specific T cells, Dr Tager for humanized mice and Dr Irvine for the development of nanoparticles for antigen delivery.